My long-term goal is to obtain a cellular and molecular understanding of the complex intercellular interactions that result in terminal differentiation of bone marrow-derived precursors into mature effector T cells. The focus of the present application is characterization of self ligands that induce positive selection in the thymus. The application is based on our recent discovery of a novel strategy of predicting and identifying self peptides promoting positive selection. The approach consists of generation of large databases of potential self peptides (made possible by the mouse genome project) and bioinformatics-based selection from these databases of small groups of candidate peptides. Selected peptides are tested for positive selection in fetal thymic organ cultures and MHC presentation of peptides is verified by screening self peptide pools by mass spectrometry analysis. In Specific aim 1 we will expand this strategy to identify multiple positively selecting peptides in four T cell receptor transgenic models. We will also experimentally test whether similarity of self peptides to antigen is a working principle of positive selection. The similarity scores of self-ligands identified by three independent means will be plotted against the ability to induce positive selection. A complementary approach will consist of generating synthetic peptide libraries of graded similarity to antigen. The ability of these libraries to induce positive selection will be determined. In the Specific Aim 2 we will determine if and how the duration of T cell receptor signaling affects positive and negative selection. Using a novel in vitro experimental delivery of positively selecting signals we will observe the kinetics of MAP kinase activation induced by negatively or positively selecting ligands. In a complementary approach we will assess the effects on positive versus negative selection of limiting the duration of signaling. In Specific aim 3 we will determine the structural basis for selective involvement of amino-terminal portion of peptide in positive selection. These findings should greatly contribute to our knowledge of the formation of functional TCR repertoire. This information will be valuable for vaccine development, immunomodulation therapy in organ transplantation, autoimmune diseases, lymphoproliferative diseases and cancer.